RT Journal Article SR Electronic(1) A1 Cooley, Richard B. A1 Dubbels, Bradley L. A1 Sayavedra-Soto, Luis A. A1 Bottomley, Peter J. A1 Arp, Daniel J.YR 2009 T1 Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’ JF Microbiology, VO 155 IS 6 SP 2086 OP 2096 DO https://doi.org/10.1099/mic.0.028175-0 PB Microbiology Society, SN 1465-2080, AB Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5 alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k cat : K m ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K m values. The low micromolar K m for linear C2–C5 alkanes and the millimolar K m for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K m and k cat values to those of methane. This inability to kinetically discriminate between the C1 alkane and C1 alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C2–C5 do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5 alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ≥C2, despite its high sequence homology with sMMO. These results represent, to the best of our knowledge, the first kinetic in vitro characterization of the closest known homologue of sMMO., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.028175-0